Mitochondrial function in Cell death in PD

Pathology Loss of SN pigmented dopamine neurons Lewy bodies Lewy neurites-multiple brain regions Lewy bodies stain with antibodies to alpha synuclein, ubiquitin, others Also present in autonomic and submucosal ganglia Clear that PD is more than just a disorder of dopamine deficiency, but that SN cells for an unknown reason are even more sensitive to the stresses of the pathological abn than other parts of the brain

Environmental factors Post-encephalitic and post-traumatic PD MPTP (meperidine analog) 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine, injected, metabolized to MPP+, taken up into dopaminergic neurons by transporter, concentrated as MPP+ in mitochondria Rotenone, paraquat

Pyruvate Acetyl CoA TCA cycle NADH H+H+ H + leak controls basal metabolic rate ADP + P i ATP Respiratory enzyme complexes NADH dehydrogenase Succinate dehydrogenase Cytochrome b CoQ Cytochrome oxidase ATP synthase Lactate Anaerobic Glycolysis FADH 2 Oligomycin X Mitochondrial energy production Inner mitochondrial membrane H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+

Mito dysfunction In PD, SN neurons accumulate mito DNA deletions at an abn rate-suggests that oxidative stress is occurring. Impaired cell respiration results from mito DNA deficiency that causes respiratory chain deficiency A mutation in the gene for mito DNA polymerase assoc. with accumulation in deletions of mito DNA, SN loss, early PD Common feature of PD is evidence of Complex 1 deficiency Complex 1 also affected by rotenone and MPTP When rotenone given chronically to rodents, it causes complex 1 deficiency, dopaminergic cell loss in SN

Mito dysfunction 6-hydroxydopamine and paraquat cause oxidative stress, mimic mito toxicity seen with MPTP Findings led to trials of coenzyme Q, vit E, creatine, all anti-oxidant and pro- mitochondrial compounds

Mitochondria in PD Contributions to understanding the pathogenesis of PD by familial inherited forms of PD

Genetic mutations-  -synuclein First to be identified was  -synuclein Point mutations caused familial PD, rare AD form Mice lacking gene for  -synuclein show resistance to MPTP-induced dopaminergic toxicity In Lewy bodies it is present in aggregated form in insoluble filaments that are hyperphosphorylated and ubiquitinated It is likely that misfolded  synuclein is toxic to neurons Factors that increase aggregation of  synuclein are genetic mutations, proteasome and mitochondrial dysfunction, oxidative stress, phosphorylation. Likely involved in synaptic vesicle function

Genetic mutations-Parkin Mutations in gene for Parkin cause aut. Recessive form of PD Most common genetic cause-50% with family history Parkin is an E3 ligase-participates in addition of ubiquitin molecules to target proteins, marking them for degradation by the proteasome Loss of parkin function therefore leads to an inability to break down toxic substances with subsequent neuronal dysfunction and cell death. Parkin substrates p38/JTV and FBP-1 accumulate in sporadic cases of PD and in Parkin K/O mice Role of ubiquitination in development of PD is a promising field of study

PINK-1 Mutations in this gene encoding PTEN (Phosphatase and tensin homologue)-induced putative kinase 1(PINK-1) cause aut. recessive PD. Mitochondrial protein kinase, substrates unknown Targets to mitochondria K/O in Drosophila assoc. with mitochondrial dysfunction, reduced respiratory chain activity, reduced mito DNA, reduced ATP content of tissues and increased propensity to apoptosis of affected cells such as muscle Parkin over-expression rescues the loss of function phenotype of PINK-1 K/O in Drosophila, Parkin downstream of PINK-1-links mitochondria to proteasome Patients with genetic mutations in Parkin or PINK-1 are clinically indistinguishable

Savitt et al., 2006

cytochrome c VDAC outer membrane inner membrane Intermembrane space BCL-2 proteins induce apoptosis by releasing cytochrome c from mitochondria caspase-9 casp a se-3 Neuronal death BAX

The mitochondrial permeability transition pore is a double membrane-spanning ion channel The mitochondrial permeability transition pore is a double membrane-spanning ion channel Outer mitochondrial membrane Inner mitochondrial membrane VDAC/BCL-xL Ca 2+ or Zn 2+ mPTP Cytochrome c VDACmPTP CyD Messenger BAD ANT

Inhibition of proteasome function may cause PD-like symptoms in animal models We injected animals with a proteasome inhibitor (PSI) After 2 week of injections, animals had –Slowness of movement –Decreased dopamine metabolites McNaught et al, Ann Neurol, 2004

Assay of mitochondrial function Can protein aggregates produce or aggravate mitochondrial dysfunction? Can the mito dysfunction cause neuronal death of sensitive neurons?

Organelle attached Patch Clamp Technique Mitochondria isolated from PSI treated rat basal ganglia, as early as one week after first PSI injection (i.e. before appearance of clinical phenotype)

rat brain homogenize low speed spin high speed spin digitonin treatment Ficoll gradient hypo-osmotic treatment Isolation of Mitochondria

Organelle attached Patch Clamp Technique

Measuring death channel activity with the mitochondrial recording technique

% activity ClosedSmallInter.Large CTL Striatum PSI Striatum *** * * CTL Cortex PSI Cortex ClosedSmallInter.Large Proteasome inhibitor injection into rats produces large conductance activity of mitochondrial membranes isolated from subcortex